Modified forms of pullulanase

ABSTRACT

The present invention relates to modified pullulanases useful in the starch industry. The present invention provides methods for producing the modified pullulanase, enzymatic compositions comprising the modified pullulanase, and methods for the saccharification of starch comprising the use of the enzymatic compositions.

RELATED APPLICATIONS

[0001] The present application is a continuation-in-part application ofU.S. application Ser. No. 09/034,630 filed Mar. 4, 1998 which isincorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to modified forms of pullulanasewhich maintain the ability to catalyze the hydrolysis of analpha-1,6-glucosidic bond, compositions which comprise the modifiedpullulanase, methods of making the modified pullulanase and methods ofusing the modified pullulanase, especially for the saccharification ofstarch.

BACKGROUND OF THE INVENTION

[0003] Starch, the essential constituents of which are linear amyloseand branched amylopectin glucose polymers can be converted into simplesugars by an enzymatic process carried out in two stages: one stage ofliquefaction of the starch and one stage of saccharification of theliquefied starch. In order to obtain a high conversion level of thestarch, pullulanase (E.C. 3.2.1.41, α-dextrin 6-glucano-hydrolase alsotermed alpha-1,6-glucosidase) has been used to catalyse the hydrolysisof alpha-1,6-glucosidic bonds.

[0004] Pullulanase enzymes in the art include those known to haveoptimum activity at acidic pH as well as those known to have activity atalkaline pH. Pullulanases described in the art include pullulanasederived from a strain of Bacillus acidopullulyticus described as havingan optimum activity at a pH of 4-5 at 60° C. (U.S. Pat. No. 4,560,651);pullulanase derived from Bacillus naganoensis described as having amaximum activity at a pH of about 5, measured at 60° C. and a maximumactivity at a temperature of about 62.5° C., measured at a pH of 4.5(U.S. Pat. No. 5,055,403); pullulanase derived from Bacillus sectorramusdescribed as having an optimum pH at 5.0 to 5.5 and an optimumtemperature at 50° C. (U.S. Pat. No. 4,902,622); and pullulanase derivedfrom Bacillus brevis PL-1 described as having activity at 4.5-5.5 at 60°C. (JP 04/023985).

[0005] Pullulanase can be used with glucoamylase or β-amylase for theproduction of high glucose and high maltose syrups. In addition toincreasing the yields of sugars, pullulanase reduces reaction time,allows high substrate concentrations and a reduction of up to 50% in theuse of glucoamylase (Bakshi et al. (1992) Biotechnology Letters vol. 14pp. 689-694).

SUMMARY OF THE INVENTION

[0006] The present invention relates to the surprising and unexpecteddiscovery by Applicants that modified forms of pullulanase retain theability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond. Thepresent invention provides modified forms of pullulanase and methods forproducing the modified pullulanase, especially in recombinant hostmicroorganisms. The present invention further relates to enzymaticcompositions comprising a modified form of pullulanase useful in thesaccharification of starch and methods for the saccharification ofstarch comprising the use of the enzymatic compositions.

[0007] The present invention is based, in part, upon the discovery thatwhen pullulanase obtained from Bacillus deramificans was recombinantlyexpressed and cultured in Bacillus licheniformis, the pullulanaseproduced was a mixture of modified forms yet the modified forms ofpullulanase surprisingly retained the ability to catalyze the hydrolysisof an alpha-1,6-glucosidic bond. The modified forms comprised B.deramificans pullulanase truncated at the amino terminus, i.e., having adeletion of amino acids from the amino terminus, and B. deramificanshaving additional amino acids at the amino terminus of the maturepullulanase. Therefore, in one aspect, the present invention providesmodified pullulanase having a deletion of amino acids from the aminoterminus of a pullulanase obtainable from a gram-positive or agram-negative microorganism as long as the modified pullulanase retainsthe ability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond.In another aspect, the present invention provides modified pullulanasehaving additional amino acids at the amino terminus of a pullulanaseobtainable from a gram-negative or gram positive microorganism as longas the modified pullulanase retains the ability to catalyze thehydrolysis of an alpha-1,6-glucosidic bond. The present invention alsoencompasses amino acid variations of a pullulanase obtainable from agram-negative or gram positive microorganism as long as the modifiedpullulanase retains the ability to catalyze the hydrolysis of analpha-1,6-glucosidic bond.

[0008] In one embodiment, the modified pullulanase is a modification ofpullulanase obtainable from Klebsiella species. In another embodiment,the modified pullulanase is a modification of pullulanase obtainablefrom Bacillus species. In yet another embodiment, the modifiedpullulanase is a modification of pullulanase obtainable from Bacillusincluding but not limited to B. subtilis, B. deramificans, B.stearothermophilus, B. naganoensis, B. flavocaldarius, B.acidopullulyticus, Bacillus sp APC-9603, B. sectorramus, B. cereus, B.fermus. In a preferred embodiment, the modified pullulanase is amodification of pullulanase obtainable from B. deramificans having thedesignation T89.117D (LMG P-13056) deposited Jun. 21, 1993 under theBudapest Treaty in the LMG culture collection, University of Ghent,Laboratory of Microbiology, K. L. Ledeganckstraat 35, B-9000 GHENT,Belgium.

[0009] In one embodiment, the modified pullulanase has a deletion ofabout 100 amino acids from the amino terminus of a pullulanase. Inanother embodiment, the modified pullulanase has a deletion of about 200amino acids from the amino terminus of a pullulanase and in yet anotherembodiment, the modified pullulanase has a deletion of about 300 aminoacids from the amino terminus of a pullulanase.

[0010] In a further embodiment, the modified pullulanase has a deletionof 98 amino acids from the amino terminus of pullulanase obtainable fromB. deramificans. In an additional embodiment, the modified pullulanasehas a deletion of about 102 amino acids from the amino terminus ofpullulanase obtainable from B. deramificans. In a further embodiment,the modified pullulanase has at least one additional amino acid at theamino terminus of pullulanase obtainable from B. deramificans. Inanother embodiment, the modified pullulanase has an additional aminoacid residue, Alanine, added to the amino terminus of pullulanaseobtainable from B. deramificans.

[0011] Modified forms of pullulanase having a decrease in molecularweight provide the advantage of higher specific activity (activity/unitweight) and therefore, less weight of pullulanase activity is requiredin a saccharification process to obtain results equivalent to the use ofa naturally occurring pullulanase obtainable from or produced by amicroorganism. The recombinant production of modified pullulanase astaught herein provides for enzymatic compositions comprising at least60% and at least 80% pullulanase activity. In one embodiment, theenzymatic composition comprises at least one modified pullulanase. Inanother embodiment, the enzymatic composition comprises more than onemodified pullulanase. Such enzymatic compositions are advantageous tothe starch processing industry due to their ability to produce a highglucose yield over a shortened saccharification time without the loss ofglucose yield associated with reversion reaction products. Furthermore,it was unexpectedly found that in using an enzymatic compositioncomprising 20% glucoamylase and 80% pullulanase, higher startingdissolved solids (DS) could be used in a saccharification process,thereby increasing production plant capacity without an increase incapital investment. Additionally, saccharification at higher dissolvedsolids increases mechanical compression capacity thereby providing for amore energy efficient process.

[0012] In one embodiment, the present invention provides modifiedpullulanase produced by the method comprising the steps of obtaining arecombinant host cell comprising nucleic acid encoding maturepullulanase, culturing said host cell under conditions suitable for theproduction of modified pullulanase and optionally recovering themodified pullulanase. In one embodiment, the host cell is Bacillus,including but not limited to B. subtilis, B. licheniformis, B. lentus,B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens,B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis. In apreferred embodiment, the Bacillus cell is B. licheniformis whichcomprises a first gene encoding the Carlsberg protease and a second geneencoding endo Glu C, the first and/or second gene which codes for theprotease(s) having been altered in the Bacillus species such that theprotease activity is essentially eliminated and the nucleic acidencoding the mature pullulanase is obtainable from B. deramificans.

[0013] In an alternative embodiment, the present invention providesmethods for the production of a modified pullulanase in a recombinanthost cell comprising the steps of obtaining a recombinant microorganismcomprising nucleic acid encoding a modified pullulanase, culturing themicroorganism under conditions suitable for the production of themodified pullulanase and optionally recovering the modified pullulanaseproduced. In one embodiment, the host cell is a gram-negative orgram-positive microorganism. In another embodiment, the host cell is aBacillus including but not limited to B. subtilis, B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillusthuringiensis. In another embodiment, the Bacillus cell is B.licheniformis which comprises a first gene encoding the Carlsbergprotease and a second gene encoding endo Glu C, the first and/or secondgene which codes for the protease(s) having been altered in the Bacillusspecies such that the protease activity is essentially eliminated andthe nucleic acid encodes a modified pullulanase that is a modificationof pullulanase obtainable from B. deramificans.

[0014] The present invention also provides nucleic acid comprising apolynucleotide sequence encoding modified pullulanase. In oneembodiment, the nucleic acid has at least 70% identity, at least 80%identity, at least 90% identity or at least 95% identity to thepolynucleotide sequence shown in SEQ ID NO: 1, which encodes pullulanaseobtainable from B. deramificans. The present invention also providesexpression vectors and host microorganisms comprising nucleic acidencoding a modified pullulanase of the present invention.

[0015] The present invention provides an enzymatic compositioncomprising at least one modified pullulanase of the present invention.In one embodiment, the enzymatic composition comprises multiple modifiedpullulanase forms. In another embodiment, the composition furthercomprises an enzyme selected from the group consisting of glucoamylase,alpha-amylase, beta-amylase, alpha-glucosidase, isoamylase,cyclomaltodextrin, glucotransferase, beta-glucanase, glucose isomerase,saccharifying enzymes, and/or enzymes which cleave glucosidic bonds. Ina preferred embodiment, the enzymatic composition comprises a modifiedpullulanase and glucoamylase. In one embodiment, the glucoamylase isderived from an Aspergillus strain. In another embodiment, theglucoamylase is derived from an Aspergillus strain including but notlimited to Aspergillus niger, Aspergillus awamori and Aspergillusfoetidus. The enzymatic composition may be in a solid form or a liquidform. In one embodiment of the present invention, the enzymaticcomposition comprises at least 60% modified pullulanase and in anotherembodiment, the composition comprises at least 80% modified pullulanase.

[0016] The present invention also provides a process for thesaccharification of starch, wherein said process allows for reducedconcentrations of saccharification reversion by-products, comprising thestep of contacting aqueous liquified starch with an enzyme compositioncomprising modified pullulanase. In one embodiment, the process furthercomprises the steps of heating said liquified starch, and recoveringproduct. In one embodiment of the process, the enzyme compositionfurther comprises glucoamylase. In another embodiment of the process,the contacting is at a pH of about less than or equal to 7.0 and greaterthan or equal to 3 and in yet another, the pH is about 4.2. In a furtherembodiment of the process said heating is at a temperature range ofbetween 55 and 65 degrees C. In another embodiment, the temperature isabout 60 degrees C.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1E illustrate the nucleic acid (SEQ ID NO:1) encodingthe mature amino acid (SEQ ID NO:2) sequence of pullulanase obtainablefrom B. deramificans.

[0018] FIGS. 2A-2D are an alignment of amino acid sequences ofpullulanase obtainable from B. deramificans (designatedpullseqsig.seq.PRO), B. subtilis (designated subpull.seq.pro), and K.pneumonia (designated klebpnseqsig.seq.pro) showing the conserveddomains and variability of the amino terminus of these pullulanases.This alignment also includes the signal sequences for the respectivepullulanases.

[0019] FIGS. 3A-3C illustrate a timecourse of fermentation and thevarious species of modified pullulanase that are formed during thefermentation. Peak 1 designates the mature B. deramificans pullulanasehaving a molecular weight of 105 kD; peak 2 designates the modifiedpullulanase which has a deletion of 102 amino acids from the aminoterminus of mature B. deramificans pullulanase; and peak 3 designatesthe modified pullulanase which has a deletion of 98 amino acids from theamino terminus as measured by standard HPLC analysis. FIG. 3Aillustrates the fermentation over 37 hours. FIG. 3B illustrates thefermentation over 60 hours. FIG. 3C illustrates the fermentation over 70hours.

[0020] FIGS. 4A-4D illustrate the stability of the modified pullulanasespecies as a function of pH as measured by standard HPLC analysis. FIG.4A illustrates the pullulanase stability at 24 hours at a pH of 4.5 atroom temperature. FIG. 4B illustrates the pullulanase stability at 24hours at a pH of 5.5 at room temperature. FIG. 4C illustrates thepullulanase stability at 24 hours at a pH of 6.5 at room temperature.FIG. 4D illustrates the pullulanase stability at 96 hours at a pH of 4.5at room temperature.

[0021] FIGS. 5A-5C illustrate the effect of enzymatic compositionscomprising various pullulanase and glucoamylase concentrations on thefinal glucose yield and disaccharide formation over saccharificationtime. The solid line refers to an enzymatic blend comprising 80%pullulanase activity (including modified pullulanase having a deletionof 98 amino acids from the amino terminus of B. deramificans; modifiedpullulanase having a deletion of 102 amino acids from the amino terminusof B. deramificans; mature B. deramificans pullulanase and mature B.deramificans pullulanase having an additional amino acid (alanine) onthe amino terminus) and 20% glucoamylase (20:80). The dotted line refersto an enzymatic composition comprising an enzyme blend comprising 75%glucoamylase obtainable from Aspergillus sp. and 25% mature pullulanaseobtainable from B. deramificans (75:25). The solid line with squaresrefers to di-saccharides formed with the enzyme blend comprising 20%glucoamylase and 80% pullulanase activity as described above (20:80)over the saccharification time and the dotted line with circles refersto the di-saccharides formed with the 75:25 over the saccharificationtime. The left X-axis is % glucose yield and the right X-axis is %di-saccharides. FIG. 5A refers to the saccharification process using0.550 liters of enzymatic composition per metric ton of dissolvedsolids; FIG. 5B refers to the saccharification process using 0.635liters of enzymatic composition per metric ton of dissolved solids; FIG.5C refers to the saccharification process using 0.718 liters ofenzymatic composition per metric ton of dissolved solids. This Figureillustrates that a 20:80 enzymatic composition is able to increase thefinal glucose yield without an increase in undesirable disaccharideformation.

[0022]FIG. 6 illustrates the effect of dissolved solids (%w/w) (Y axis)on the final glucose yield during saccharification of liquefied starchusing enzyme compositions 20:80, 75:25, and 100% glucoamylase at 0.55liters of enzyme per metric ton of dissolved solids. Line A is theenzymatic composition 20:80 described in FIGS. 5A-5C; line B is theenzymatic composition 75:25 and line C is an enzymatic compositioncomprising 100% glucoamylase.

DETAILED DESCRIPTION

[0023] Definitions

[0024] The term pullulanase as used herein refers to any enzyme havingthe ability to cleave the alpha-1,6 glucoside bond in starch to producestraight chain amyloses. These enzymes are preferably classified in EC3.2.1.41 and include neopullulanases.

[0025] As shown in FIGS. 2A-2D, there are regions of similarity amongpullulanases obtainable from gram positive and gram negativemicroorganisms. The amino acid alignment of pullulanase obtainable fromBacillus deramificans with pullulanase obtainable from K. pneumonia andB. subtilis reveals that when the conserved domains are aligned, theamino terminus not associated with the conserved domains is of varyinglength. As used herein, the term “modified” when referring topullulanase means a pullulanase enzyme in which the conserved domainsare retained while any length of amino acids in the amino terminusportion of the naturally occurring amino acid sequence not associatedwith the conserved domains has been altered by a deletion of these aminoacid residues or by addition of at least one amino acid to the aminoterminus as long as the modified pullulanase retains the ability tocatalyze the hydrolysis of an alpha-1,6-glucosidic bond. The deletion inthe amino terminal amino acids of a pullulanase can be of varyinglength, but is at least three amino acids in length and the deletion cango no further than the beginning of the first conserved domain which inB. deramificans is the tyrosine at amino acid residue 310 as shown inFIGS. 1A-1E. In one embodiment, the deletion is about 100 amino acidsfrom the amino terminus of the mature pullulanase. In anotherembodiment, the deletion is about 200 amino acids from the aminoterminus of the mature pullulanase and in another embodiment, thedeletion is about 300 amino acids from the amino terminus of the maturepullulanase. In a preferred embodiment, the modification is a deletionof 98 amino acids from the amino terminus of B. deramificans. In yetanother embodiment, the deletion is 102 amino acids from the aminoterminus of B. deramificans. In a further embodiment, the modificationis an addition of at least one amino acid to the amino terminus of thenaturally occurring mature pullulanase obtainable from Bacillusderamificans. In another preferred embodiment, the amino acid residue,Alanine, is added to the amino terminus of the mature pullulanase. Asused herein the term “mature” refers to a protein which includes theN-terminal amino acid residue found after the natural cleavage site ofthe signal sequence.

[0026] As illustrated in FIGS. 2A-2D, B. deramificans pullulanase and K.pneumonia pullulanase are examples of pullulanases having similaritiesin the length of the amino terminus up to the beginning of the firstconserved domain (which in B. deramificans is amino acid residue 310Tyrosine). B. subtilis pullulanase is an example of a pullulanase havinga shorter length of amino acid residues up to the beginning of the firstconserved domain as shown in FIG. 2B.

[0027] As used herein, “nucleic acid” refers to a nucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. The present invention encompasses polynucleotideshaving at least 70%, at least 80%, at least 90% and at least 95%identity to the polynucleotide encoding B. deramificans pullulanase, aswell as polynucleotides encoding a pullulanase activity capable ofhybridizing to nucleic acid encoding B. deramificans pullulanase underconditions of high stringency.

[0028] The terms “isolated” or “purified” as used herein refer to anucleic acid or amino acid that is removed from at least one componentwith which it is naturally associated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention relates to the discovery that pullulanaserecombinantly produced in a Bacillus host is modified yet unexpectedlyretains the ability to catalyze the hydrolysis of analpha-1,6-glucosidic bond. The modification of the pullulanase productrecombinantly produced appears to be a result of misprocessing of thesignal sequence by a signal peptidase as well as susceptibility toextracellular proteolytic processing. The modified pullulanase is usedto produce compositions and methods useful in the starch industry.

[0030] I. Pullulanase Sequences

[0031] The present invention encompasses any modified pullulanase whichretains the ability to catalyze the hydrolysis of analpha-1,6-glucosidic bond. A variety of pullulanases have been describedin the art, including those obtainable from or naturally produced bygram-positive microorganisms as well as gram-negative microorganisms.Microorganisms which naturally produce pullulanase include, but are notlimited to, B. deramificans (having the designation T89.117D in the LMGculture collection, University of Ghent, Laboratory of Microbiology-K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium) the nucleic acid (SEQ IDNO:1) and amino acid (SEQ ID NO:2) sequence being disclosed in FIGS.1A-1E; B. naganoensis (American Type Culture Collection, ATCC accessionnumber 53909), disclosed in U.S. Pat. No. 5,056,403 issued Oct. 8, 1991;B. acidopullulyticus (National Collection of Industrial Bacteria, TorryResearch Station, Aberdeen, Scotland, NCIB 11607, NCIB 11610, NCIB11611, NCIB 11636, NCIB 11637, NCIB 11639, NCIB 11638, NCIB 11647, NCIB11777), disclosed in U.S. Pat. No. 4,560,651, issued Dec. 24, 1985; B.sectorramus (Fermentation Research Institute, Agency of IndustrialScience and Technology, 1-3, Higashi 1-chome, Yatabe-machi, Tsukuba-gun,Ibaraki 305 Japan FERM BP-1471), disclosed in U.S. Pat. No. 4,902,622,issued Feb. 20, 1998; Bacillus FERM BP-4204 disclosed in U.S. Pat. No.5,387,516 issued Feb. 7, 1995; B. stearothermophilus (SWISS-PROT idNEPU_BACST ac P38940); B. cereus var. mycoides (IFO 300) described in Y.Takasaki et al., 1976, Agric. Biol. Chem. 40:1515; B. fermus (IFO 3330);Klebsiella pneumonia, U.S. Pat. No. 3,897,305 (SWISS-PROT id PULA_KLEPNac P07206 and ATCC 15050; Klebsiella aerogenes (SWISS-PROT id PULA_KLEAEac P07811); Thermoanaerobium brockii (ATCC No. 33075), U.S. Pat. No.4,628,028; Streptomyces sp. described in M. Yagisawa et al., 1972, J.Ferment. Technolo. 50:572; Caldicellulosiruptor saccharolyticusdisclosed in Albertson et al., 1997, Biochimica et Biophysica Acta1354:35-39; Eschericia intermedia Ueda et al., 1967, AppliedMicrobiology vol 15:492 U.S. Pat. No. 3,716,455 (issued 1973)Streptococcus mites Walker 1968, Biochem. J., vol. 108:33; Streptomyces(Ueda et al., 1971, J. Ferment. Tech. Vol. 49: 552); Flavochromogenes,as described in U.S. Pat. No. 4,902,622; Flavobacterium esteromaticumJapanese Patent Application Kokoku 18826/1973; Cytophaga U.S. Pat. No.3,790,446 issued 1974; Lactobacillus, Micrococcus, Nocardia,Staphylococcus, Azotobactger, Sarcina England patent 11260418, U.S. Pat.No. 3,827940 issued 1974; and Actinomycetes U.S. Pat. No. 3,741,873issued 1973. Any pullulanase known in the art which comprises theconserved pullulanase regions as shown in FIGS. 2A-2D can be modified tohave deletions or additions to the amino terminus as long as themodified pullulanase retains the ability to catalyse the hydrolysis ofan alpha-1,6-glucosidic bond.

[0032] A nucleic acid sequence encoding a pullulanase can be obtainedfrom a microorganism through hybridization technology using the nucleicacid sequences that encode the conserved domains of pullulanases (asshown in FIGS. 2A-2D) as primers and/or probes. (U.S. Pat. No.5,514,576; Southern, E. 1979, Methods Enzymol. 68:152-176; Saiki, et al.1988, Science 239:487-491). In one embodiment disclosed herein for B.deramificans pullulanase, the naturally occurring nucleic acid (SEQ IDNO:1) encoding a mature pullulanase was introduced into B. licheniformishaving a deletion of the Carlsburg protease (Jacobs et al., 1985,Nucleic Acid Research 13:8913-8926) and endoGluC proteases (Kakudo etal., 1992, Journal of Bio. Chem. Vol. 267:23782-23788), the B.licheniformis comprising the nucleic acid encoding the maturepullulanase was cultured under conditions suitable for expression ofsaid nucleic acid and secretion of the expressed pullulanase. Theprotease deletions in B. licheniformis were made through techniquesknown to those of skill in the art. Through the fermentation process,the expressed pullulanase was cleaved extracellularly into multiplepullulanase species which retain the ability to catalyse the hydrolysisof alpha-1,6-glucosidic bonds. The multiple species are a pullulanasehaving a deletion of the first 98 amino acid residues from the aminoterminus and starting at glutamic acid, a pullulanase having a deletionof the first 102 amino acid residues from the amino terminus (andstarting at glutamic acid), and a pullulanase having the addition of atleast one amino acid residue to the amino terminus of the maturepullulanase, along with the mature pullulanase as shown in FIGS. 1A-1E.As shown in Example II, it appears that the extracellular cleaving intomultiple species may be due to a protease activity in the fermentationbroth.

[0033] In an alternate embodiment of the present invention, nucleic acidencoding a mature pullulanase is genetically engineered to create amodified pullulanase having a deletion of amino acids at the aminoterminus or having amino acids added at the amino terminus. Thegenetically engineered pullulanase is introduced into a host cell,preferably a Bacillus host cell, and cultured under conditions suitablefor expression and secretion of the modified pullulanase. Nucleic acidencoding a mature pullulanase can be a naturally occurring sequence, avariant form of the nucleic acid or from any source, whether natural,synthetic, or recombinant.

[0034] Regional sequence homologies in starch degrading enzymes havebeen disclosed in Janse et al. (1993) Curr. Genet. 24:400-407. Jansedisclose the conserved regions in α-amylases that are implicated insubstrate binding, catalysis, and calcium binding. An amino acidalignment of B. deramificans, B. subtilis and K. pneumonia pullulanasesis shown in FIGS. 2A-2D.

[0035] When homologies were compared in starch degrading enzymes byJanse et al., four conserved regions where noted, Regions 1, 2, 3, and4. Three of these regions were associated with specific functions foundin starch-degrading enzymes: region 1: DWINH; region 2: GFRLDMKH; andregion 4: FVDVHD. Further analysis of five Type I pullulanase sequencesby Albertson et al.(1997, Biochimica et Biophysica Acta 1354:35-39)revealed other conserved regions among a group of gram-positive andgram-negative pullulanases. These include regions called DPY, A, B, C,D, E, and YNWGY. Two regions, DPY and YNWGY were identified as beingcharacteristic of true pullulanases. Conserved regions A-E align closelywith β-sheet elements as defined for amylases. In addition, two otherconserved regions closer to the N-terminus of the pullulanase, referredto as Y and VWAP in FIGS. 2A-2D, indicate the limits of amino acidtruncations in the N-terminal of pullulanases in general. Thisprediction is based on the lack of further conserved regions of identityamong the known pullulanases beyond the Y region as one proceeds to theN-terminus. Due to the size heterogeneity of the known pullulanases, theN-terminal regions beyond the Y region call vary between approximately100-300 amino acids. For the B. deramificans pullulanase, a truncationof 309 residues would leave the first conserved region (Y at amino acidresidue 310 in FIGS. 1A-1E) intact.

[0036]B. deramificans Pullulanase

[0037] Mature B. deramificans pullulanase comprises the amino acidsequence (SEQ ID NO: 2) shown in FIGS. 1A-1E. The following descriptionof characteristics refers to mature B. deramificans pullulanase. B.deramificans pullulanase has an isoelectric point of between 4.1 and4.5, is heat stable and active in a wide temperature range. The B.deramificans pullulanase is active at an acid pH. This pullulanase iscapable of catalyzing the hydrolysis of α-1,6-glucosidic bonds presentboth in amylopectin and in pullulan. It breaks down pullulan intomaltrotriose and amylopectin into amylose. The polysaccharide pullulan,which is a polymer of maltotriose units connected to each other byalpha-1,6-linkages can be obtained from Aureobasidium pullulans(Pullaria pullulans) by the procedure of Ueda et al., AppliedMicrobiology, 11, 211-215 1963).

[0038]B. deramificans pullulanase hydrolyses amylopectin to formoligosaccharides (maltooligosaccharides). During this hydrolysis, theformation of oligosaccharides made up of about 13 glucose units (degreeof polymerization of 13, this molecule is also called “chain A”) isobserved, followed by the formation of oligosaccharides made up of about47 glucose units (degree of polymerization of 47, this molecule is alsocalled “chain B’).

[0039] The oligosaccharides with chains A and B are defined withreference to D. J. MANNERS (“Structural Analysis of Starch components byDebranching Enzymes” in “New Approaches to research on CerealCarbohydrates”, Amsterdam, 1985, pages 45-54) and B. E. ENEVOLDSEN(“Aspects of the fine structure of starch” in “New Approaches toresearch on Cereal Carbohydrates”, Amsterdam, 1985, pages 55-60).

[0040] The B. deramificans pullulanase hydrolyses potato amylopectin.This hydrolysis can be carried out with an aqueous suspension ofamylopectin in the presence of the pullulanase under the conditions ofoptimum activity of the pullulanase, that is to say at a temperature ofabout 60° C. and at a pH of about 4.3.

[0041] The B. deramificans pullulanase catalyses the condensationreaction of maltose to form tetraholosides (oligosaccharides having 4glucose units).

[0042] The B. deramificans pullulanase has a half-life of about 55hours, measured at a temperature of about 60° C. in a solution bufferedat a pH of about 4.5 and in the absence of substrate.

[0043] Half-life means that the pullulanase shows a relative enzymaticactivity of at least 50%, measured after an incubation of 55 hours at atemperature of about 60° C. in a solution buffered at a pH of about 4.5and in the absence of substrate.

[0044] The B. deramificans pullulanase is heat stable at an acid pH andshows a relative enzymatic activity of at least 55%, measured after anincubation of 40 hours at a temperature of 60° C. in a solution bufferedat a pH of about 4.5 and in the absence of substrate. It shows arelative enzymatic activity of at least 70%, measured after anincubation of 24 hours under these same conditions.

[0045] Relative enzymatic activity means the ratio between the enzymaticactivity measured in the course of a test carried out under the givenpH, temperature, substrate and duration conditions, and the maximumenzymatic activity measured in the course of this same test, theenzymatic activity being measured starting from the hydrolysis ofpullulane and the maximum enzymatic activity being fixed arbitrarily atthe value of 100.

[0046] The B. deramificans pullulanase is furthermore stable in a widerange of acid pH values. Under the conditions described below, it isactive at a pH greater than or equal to 3. In fact, the B. deramificanspullulanase shows a relative enzymatic activity of at least 85%,measured after an incubation of 60 minutes at a temperature of about 60°C. in the absence of substrate and in a pH range greater than or equalto about 3.5.

[0047] Under the conditions described below, it is active at a pH ofless than or equal to 7. In fact, the B. deramificans pullulanase showsa relative enzymatic activity of at least 85%, measured after anincubation of 60 minutes at a temperature of about 60° C. in the absenceof substrate and in a pH range less than or equal to about 5.8.

[0048] It preferably shows a relative enzymatic activity of greater than90%, measured in a pH range of between about 3.8 and about 5 under thesesame conditions.

[0049] The B. deramificans pullulanase develops an optimum enzymaticactivity, measured at a temperature of about 60° C., in a pH rangegreater than 4.0. It develops an optimum enzymatic activity, measured ata temperature of about 60° C., in a pH range less than 4.8. The B.deramificans pullulanase preferably develops an optimum enzymaticactivity, measured at a temperature of about 60° C., at a pH of about4.3. Furthermore, it develops an optimum enzymatic activity, measured ata pH of about 4.3, in a temperature range of between 55 and 65° C., andmore particularly at 60° C.

[0050] The B. deramificans pullulanase develops an enzymatic activity ofmore than 80% of the maximum enzymatic activity (the maximum enzymaticactivity being measured at a temperature of 60° C. and at a pH of 4.3)in a pH range between about 3.8 and about 4.9 at a temperature of about60° C.

[0051] The strain Bacillus deramificans T 89.117D has been deposited inthe collection called BELGIAN CORRDINATED COLLECTIONS OF MICROORGANISM(LMG culture collection, University of Ghent, Laboratory ofMicrobiology—K. L. Ledeganckstraat 35, B-9000 GHENT, Belgium) inaccordance with the Treaty of Budapest under number LMG P-13056 on Jun.21, 1993.

[0052] II. Expression Systems

[0053] The present invention provides host cells, expression methods andsystems for the production and secretion of modified pullulanase ingram-positive microorganisms and gram-negative microorganisms. In oneembodiment, a host cell is genetically engineered to comprise nucleicacid encoding a modified pullulanase. In another embodiment, the hostcell is genetically engineered to comprise nucleic acid encoding a fulllength or mature pullulanase, which upon culturing produces a modifiedpullulanase. In a preferred embodiment, the host cell is a member of thegenus Bacillus which has been modified to have a mutation or deletion ofendogenous proteases.

[0054] Inactivation of a Protease in a Host Cell

[0055] Producing an expression host cell incapable of producing anaturally occurring protease necessitates the replacement and/orinactivation of the naturally occurring gene from the genome of the hostcell. In a preferred embodiment, the mutation is a non-revertingmutation.

[0056] One method for mutating nucleic acid encoding a protease is toclone the nucleic acid or part thereof, modify the nucleic acid by sitedirected mutagenesis and reintroduce the mutated nucleic acid into thecell on a plasmid. By homologous recombination, the mutated gene may beintroduced into the chromosome. In the parent host cell, the result isthat the naturally occurring-nucleic acid and the mutated nucleic acidare located in tandem on the chromosome. After a second recombination,the modified sequence is left in the chromosome having therebyeffectively introduced the mutation into the chromosomal gene forprogeny of the parent host cell.

[0057] Another method for inactivating the protease proteolytic activityis through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoded metallo-protease gene. In another preferred embodiment,nucleic acid encoding the catalytic amino acid residues are deleted.

[0058] Deletion of the naturally occurring microorganism protease can becarried out as follows. A protease gene including its 5′ and 3′ regionsis isolated and inserted into a cloning vector. The coding region of theprotease gene is deleted from the vector in vitro, leaving behind asufficient amount of the 5′ and 3′ flanking sequences to provide forhomologous recombination with the naturally occurring gene in the parenthost cell. The vector is then transformed into the host cell. The vectorintegrates into the chromosome via homologous recombination in theflanking regions. This method leads to a strain in which the proteasegene has been deleted.

[0059] The vector used in an integration method is preferably a plasmid.A selectable marker may be included to allow for ease of identificationof desired recombinant microorgansims. Additionally, as will beappreciated by one of skill in the art, the vector is preferably onewhich can be selectively integrated into the chromosome. This can beachieved by introducing an inducible origin of replication, for example,a temperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

[0060] Integration by the Campbell-type mechanism can take place in the5′ flanking region of the protease gene, resulting in a proteasepositive strain carrying the entire plasmid vector in the chromosome inthe pullulanase locus. Since illegitimate recombination will givedifferent results it will be necessary to determine whether the completegene has been deleted, such as through nucleic acid sequencing orrestriction maps.

[0061] Another method of inactivating the naturally occurring proteasegene is to mutagenize the chromosomal gene copy by transforming amicroorganism with oligonucleotides which are mutagenic. Alternatively,the chromosomal protease gene can be replaced with a mutant gene byhomologous recombination.

[0062] The present invention encompasses Bacillus host cells havingprotease deletions or mutations, such as deletions or mutations in apr,npr, epr, mpr, isp and/or bpf and/or others known to those of skill inthe art. Disclosure relating to deleting protease(s) in thegram-positive microorganism, Bacillus, can be found in U.S. patentapplication Ser. Nos. 5,264,366; 5,585,253; 5,620,880 and EuropeanPatent No. EP 0369 817 B1

[0063] One assay for the detection of mutants involves growing theBacillus host cell on medium containing a protease substrate andmeasuring the appearance or lack thereof, of a zone of clearing or haloaround the colonies. Host cells which have an inactive protease willexhibit little or no halo around the colonies.

[0064] III. Production of Modified Pullulanase

[0065] For production of modified pullulanase in a host cell, anexpression vector comprising at least one copy of nucleic acid encodinga modified pullulanase, and preferably comprising multiple copies, istransformed into the host cell under conditions suitable for expressionof the modified pullulanase. In accordance with the present invention,polynucleotides which encode a modified pullulanase, or fusion proteinsor polynucleotide homolog sequences that encode amino acid variants ofmodified pullulanase (as long as the variant retains the ability tocatalyse the hydrolysis of a α-1,6-glucosidic bond), may be used togenerate recombinant DNA molecules that direct their expression in hostcells. A host cell may be a gram-positive or a gram-negative cell. Inone embodiment, the host cell belongs to the genus Bacillus. In anotherembodiment, the Bacillus host cell includes B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and Bacillus thuringiensis. In a preferred embodiment, the grampositive host cell is Bacillus licheniformis.

[0066] As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

[0067] Altered pullulanase polynucleotide sequences which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalentmodified pullulanase. As used herein a “deletion” is defined as a changein either nucleotide or amino acid sequence in which one or morenucleotides or amino acid residues, respectively, are absent.

[0068] As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respectively, ascompared to the naturally occurring modified pullulanase.

[0069] As used herein “substitution” results from the replacement of oneor more nucleotides or amino acids by different nucleotides or aminoacids, respectively.

[0070] The encoded protein may also show deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent modified pullulanase. Deliberateamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains theability to modulate secretion. For example, negatively charged aminoacids include aspartic acid and glutamic acid; positively charged aminoacids include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine, valine; glycine, alanine; asparagine, glutamine; serine,threonine, phenylalanine, and tyrosine.

[0071] The polynucleotides encoding a modified pullulanase of thepresent invention may be engineered in order to modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, eg,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns or to change codon preference, for example.

[0072] In one embodiment of the present invention, a polynucleotideencoding a modified pullulanase may be ligated to a heterologoussequence to encode a fusion protein. A fusion protein may also beengineered to contain a cleavage site located between the modifiedpullulanase nucleotide sequence and the heterologous protein ssequence,so that the modified pullulanase may be cleaved and purified away fromthe heterologous fusion partner.

[0073] IV. Vector Sequences

[0074] Expression vectors used in expressing the pullulanases of thepresent invention in host microorganisms comprise at least one promoterassociated with a modified pullulanase, which promoter is functional inthe host cell. In one embodiment of the present invention, the promoteris the wild-type promoter for the selected pullulanase and in anotherembodiment of the present invention, the promoter is heterologous to thepullulanase, but still functional in the host cell. In one embodiment ofthe present invention, nucleic acid encoding the modified pullulanase isstably integrated into the microorganism genome.

[0075] In a preferred embodiment, the expression vector contains amultiple cloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in the hostmicroorganism which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

[0076] V. Transformation

[0077] A variety of host cells can be used for the production ofmodified pullulanase including bacterial, fungal, mammalian and insectscells. General transformation procedures are taught in Current ProtocolsIn Molecular Biology (vol. 1, edited by Ausubel et al., John Wiley &Sons, Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, publishedMay 26, 1995).

[0078] In a preferred embodiment, the host cell is a gram-positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In a further preferred embodiment the Bacillus host isBacillus licheniformis. In one embodiment of the present invention,nucleic acid encoding a modified pullulanase of the present invention isintroduced into a host cell via an expression vector capable ofreplicating within the Bacillus host cell. Suitable replicating plasmidsfor Bacillus are described in Molecular Biological Methods for Bacillus,Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby expresslyincorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

[0079] In another embodiment, nucleic acid encoding a modifiedpullulanase of the present invention is stably integrated into the hostmicroorganism genome. Preferred host cells are gram-positive host cells.Another preferred host is Bacillus. Bacillus host cells include B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and Bacillus thuringiensis. A preferred host is Bacillussubtilis. Another preferred host is B. licheniformis. Several strategieshave been described in the literature for the direct cloning of DNA inBacillus. Plasmid marker rescue transformation involves the uptake of adonor plasmid by competent cells carrying a partially homologousresident plasmid (Contente et al., Plasmid 2:555-571 (1979); Haima etal., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J.Bacteriol. 154(3):1077-1087 (1983); and Weinrauch et al., J. Bacteriol.169(3):1205-1211 (1987)). The incoming donor plasmid recombines with thehomologous region of the resident “helper” plasmid in a process thatmimics chromosomal transformation.

[0080] Transformation by protoplast transformation is described for B.subtilis in Chang and Cohen, (1979) Mol. Gen. Genet 168:111-115; for B.megaterium in Vorobjeva et al., (1980) FEMS Microbiol. Letters7:261-263; for B. amyloliquefaciens in Smith et al., (1986) Appl. andEnv. Microbiol. 51:634; for B. thuringiensis in Fisher et al., (1981)Arch. Microbiol. 139:213-217; for B. sphaericus in McDonald (1984) J.Gen. Microbiol. 130:203; and B. larvae in Bakhiet et al., (1985, Appl.Environ. Microbiol. 49:577). Mann et al., (1986, Current Microbiol.13:131-135) report on transformation of Bacillus protoplasts andHolubova, (1985) Folia Microbiol. 30:97) disclose methods forintroducing DNA into protoplasts using DNA containing liposomes.

[0081] VI. Identification of Transformants

[0082] Whether a host cell has been transformed with a modified or anaturally occurring gene encoding a pullulanase activity, detection ofthe presence/absence of marker gene expression can suggest whether thegene of interest is present However, its expression should be confirmed.For example, if the nucleic acid encoding a modified pullulanase isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the pullulanase under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the pullulanase as well.

[0083] Alternatively, host cells which contain the coding sequence for amodified pullulanase and express the protein may be identified by avariety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridization and protein bioassay or immunoassay techniques whichinclude membrane-based, solution-based, or chip-based technologies forthe detection and/or quantification of the nucleic acid or protein.

[0084] The presence of the pullulanase polynucleotide sequence in a hostmicroorganism can be detected by DNA-DNA or DNA-RNA hybridization oramplification using probes, portions or fragments of the pullulanasepolynucleotide sequences.

[0085] VII. Assay of Pullulanase Activity

[0086] There are various assays known to those of skill in the art fordetecting and measuring pullulanase activity. An enzymatic unit of B.deramificans pullulanase (PUN) is defined as the amount of enzyme which,at a pH of 4.5, at a temperature of 60 degrees C. and in the presence ofpullulane, catalyses the release of reducing sugars at a rate of 1 μMglucose equivalent per minute.

[0087] Pullulanase activity can be measured in the presence or theabsence of substrate. In one aspect, pullulanase activity can bemeasured in the presence of substrate according to the followingprotocol. 1 ml of a 1% strength solution of pullulane in a 50 nM acetatebuffer at pH 4.5 is incubated at 60° C. for 10 minutes. 0.1 ml of asolution of pullulanase corresponding to an activity of between 0.2 and1 PUN/ml is added thereto. The reaction is stopped after 15 minutes byaddition of 0.4 ml of 0.5 M NaOH. The reducing sugars released areanalyzed by the method of SOMOGYI-NELSON [J. Biol. Chem., 153 (1944)pages 375-380; and J. Biol. Chem., 160 (1945), pages 61-68].

[0088] Another method can be used to analyze the pullulanase. Theenzymatic reaction in the presence of pullulane is carried out inaccordance with the above test conditions, and is then stopped byaddition of sulphuric acid (0.1 N). The hydrolysis products of pullulaneare then subjected to HPLC chromatography (HPX-87H column from BIO-RAD;the mobile phase is 10 mM H₂SO₄) in order to separate the variousconstituents. The amount of Maltotriose formed is estimated bymeasurement of the area of the peak obtained.

[0089] The so-called debranching activity, that is to say the hydrolysisof the α-1,6-glucosidic bonds present in amylopectin, can be quantifiedby the increase in the blue coloration caused, in the presence ofiodine, by the release of amylose from amylopectin. The debranchingenzymatic activity is measured in accordance with the followingprotocol. 0.4 ml of a 1% strength amylopectin solution containing a 50mM acetate buffer at pH 4.5 is incubated at 60° C. for 10 minutes. Thereaction is initiated by addition of 0.2 ml of pullulanase, and isstopped after 30 minutes by addition of 0.4 ml of 0.3 M HC1. 0.8 ml of a0.0025% (v/v) strength solution of iodine is then added to 0.2 ml ofthis reaction mixture and the optical density is measured at 565 nm.

[0090] A preferred method is disclosed in Example IV and relies on acolorimetric method that utilizes a soluble red-pullulan substrate forthe determination of pullulanase activity. As the pullulanase enzymehydrolyzes the substrate, soluble fragments of the dyed substrate arereleased into the reaction solution. The substrate is then precipitatedwith an ethanol solution and the supernatant is evaluated for colorintensity with spectrophotometer. In this assay, the degree of colorintensity is proportional to the enzyme activity.

[0091] VIII. Secretion of Recombinant Proteins

[0092] Means for determining the levels of secretion of a modifiedpullulanase in a host microorganism and detecting secreted proteinsinclude, using either polyclonal or monoclonal antibodies specific forthe protein. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul Minn.) and Maddox D E et al (1983, J Exp Med 158:1211).

[0093] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

[0094] A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 and incorporated herein by reference.

[0095] IX. Purification of Proteins

[0096] Host cells transformed with polynucleotide sequences encodingmodified pullulanase may be cultured under conditions suitable for theexpression and recovery of the pullulanase from cell culture. Theprotein produced by a recombinant gram-positive host cell comprising amutation or deletion of endogenous protease activity will be secretedinto the culture media. Other recombinant constructions may join themodified pullulanase polynucleotide sequences to a nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53).

[0097] Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath J (1992)Protein Expr Purif 3:263-281), protein A domains that allow purificationon immobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and the heterologous protein can be used to facilitatepurification.

[0098] X. Uses of the Present Invention

[0099] Modified pullulanase

[0100] A modified pullulanase of the present invention finds use invarious industries including the food industry, the pharmaceuticalsindustry and the chemical industry. A modified pullulanase can be usedin baking as an “anti-staling” agent, that is to say as an additive toprevent bread becoming stale during storage, or in brewing duringproduction of low-calorie beers. The pullulanase can also be used in thepreparation of low-calorie foods in which amylose is used as asubstitute for fats. The pullulanase can be used, for example, toclarify fruit juices.

[0101] For food applications, the pullulanase can be immobilized on asupport. The techniques for immobilization of enzymes are well known tothe expert.

[0102] The pullulanase can also be used to hydrolyse amylopectin and toform oligosaccharides starting from this amylopectin. The pullulanasecan also be used to form tetraholosides starting from maltose.

[0103] The pullulanase can also be used to condense mono- oroligo-saccharides, creating bonds of the alpha-1,6 type. The pullulanasecan be used for liquefaction of starch.

[0104] A modified pullulanase can be used in the same manner as itsrespective unmodified form. A modified pullulanase, which in unmodifiedform has activity under alkaline conditions, will retain activity underalkaline conditions. A modified pullulanase which in unmodified form hasactivity under acidic conditions, will retain activity under acidicconditions. A particular modified pullulanase will be formulatedaccording to the intended uses. Stabilizers or preservatives can also beadded to the enzymatic compositions comprising a modified pullulanase.For example, a modified pullulanase can be stabilized by addition ofpropylene glycol, ethylene glycol, glycerol, starch, pullulane, a sugar,such as glucose or sorbitol, a salt, such as sodium chloride, calciumchloride, potassium sorbate, and sodium benzoate, or a mixture of two ormore of these products. The enzymatic compositions according to theinvention can also comprise one or more other enzymes. Such enzymesinclude but are not limited to glucoamylase, alpha-amylase,beta-amylase, alpha-glucosidase, isoamylase, cyclomaltodextrin,glucotransferase, beta-glucanase, glucose isomerase, saccharifyingenzymes, and enzymes which cleave glucosidic bonds or a mixture of twoor more of these. In a preferred embodiment, the enzymatic compositioncomprises a modified pullulanase of the present invention at 80% and aglucoamylase at 20%.

[0105] The manner and method of carrying out the present invention maybe more fully understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto. All references and patent publications disclosed herein arehereby incorporated by reference.

EXAMPLES Example I

[0106] Example I illustrates the production of a modified pullulanase asdescribed herein. The nucleic acid sequence encoding a pullulanase ismodified by recombinant DNA techniques such as standard PCRprimer-directed enzymatic amplification of DNA with a thermostable DNApolymerase. (Saiki, R. K., et al., 1988, Science 239:487-491.) and PCRfusion techniques (Fleming, A. B., et al. Appl. Environ. Microbiol 61,3775-3780). DNA encoding the desired modified pullulanase is fused tothe C-terminus of a signal sequence, preferably a host microorganismsignal sequence. This construct is cloned and transformed into a hostcell, such as, B. subtilis or B. licheniformis, and cultured understandard fermentation conditions. The modified pullulanase is purifiedfrom the fermentation broth and assayed for activity.

Example II

[0107] Example II describes the modified forms of pullulanase obtainedupon culturing the recombinant B. licheniformis host cell comprisingnucleic acid encoding a mature B. deramificans pullulanase wherein thehost cell has a deletion of the Carlsburg and endo GluC proteases. TheB. licheniformis was cultured under standard fermentation conditions ina complex media. The fermentation broth was subjected to standard HPCLanalysis and the results are shown in FIGS. 3A-3C which illustrate atimecourse of the various species of modified pullulanase formed duringthe fermentation process. Peak 1 designates the mature B. deramificanspullulanase having a molecular weight of 105 kD; peak 2 designates themodified pullulanase which has deletion of 102 amino acids from theamino terminus of mature B. deramificans pullulanase; and peak 3designates the modified pullulanase which has a deletion of 98 aminoacids from the amino terminus as measured by standard HPLC analysis. Themodified pullulanase species which has an additional amino acid on themature sequence is not detectable by HPLC analysis but was detected uponnucleic acid sequencing. FIGS. 3A-3C illustrate that over fermentationtime, Peak 1 corresponding to the mature B. deramificans pullulanasedecreases while Peaks 2 and 3 increase. FIGS. 4A-4D illustrate thestability of the modified pullulanase produced upon fermentation of B.licheniformis having a deletion of the Carlsburg and endoGluC proteases.B. licheniformis comprising nucleic acid encoding a mature B.deramificans was cultured under conditions suitable for the expressionand secretion of the modified pullulanase and the fermentation broth wasadjusted to a pH of 4.5, 5.5 and 6.5 at room temperature. The modifiedpullulanase was most stable at a pH of 4.5.

Example III

[0108] Example III describes the saccharification process comparingenzymatic compositions comprising different percentages of pullulanase.Enzymatic compositions comprising either 20% glucoamylase:80% modifiedpullulanase (20:80) activity or 75% glucoamylase:25% pullulanaseactivity (75:25) were tested in saccharification processes at aconcentration of 0.550, 0.635 and 0.718 liters of enzymatic compositionper metric ton of dissolved solids. As shown in FIGS. 5A-5C, anenzymatic composition comprising 20% glucoamylase and 80% pullulanaseactivity is able to increase the final glucose yield without an increasein undesirable disaccharide formation. Furthermore, the absoluteconcentration of the 20:80 enzyme composition can be increased withoutthe undesirable increase in disaccharide formation that is seen with the75:25 enzyme composition or glucoamylase alone.

Example IV

[0109] Example IV describes an assay for the determination of activityof a modified pullulanase of the present invention. This assay is basedon a colorimetric method that utilizes a soluble red-pullulan substratefor the determination of pullulanase activity.

[0110] Reagent Preparation

[0111] A 200 mM Sodium Acetate buffer pH 5.0 w/Acarbose (density˜1.010)was prepared by weighing out 16.406 g of anhydrous Sodium acetate or27.21 g of Sodium acetate trihydrate and dissolving it in 900 mis ofdeionized water (DI) in 1 L graduated cylinder by stirring with amagnetic stir bar. The pH was adjusted to 5.0 with glacial acetic acid.0.300 g of Acarbose was added to the solution and allowed to dissolve.The volume was brought up to 1000 mL with DI water and mixed.

[0112] 2% Red Pullulan Substrate Preparation

[0113] 1.00 g of Red Pullulan substrate was weighed out and dissolved in50 mL of sodium acetate buffer by stirring with a magnetic stir bar forapproximately 20-30 minutes. This solution is stable for two weeksstored at 4° C.

[0114] Preparation of a Working Standard

[0115] Using positive displacement pipettes a 1:10 dilution of thePullulanase Standard was prepared. The assigned activity of the standardwas 195.9 ASPU/ml. The following working concentrations were preparedfrom the standard from the 1:10 stock dilution.

[0116] Sample Preparation

[0117] For a control, Optimax L-300 MA7EC191 PU B13-19A available fromGenencor International was used. The control was diluted 1:1000 insodium acetate buffer. All samples were diluted in sodium acetate bufferto obtain final reaction absorbances that fall on the calibration curve.The sample was brought to room temperature. A minimum of 100 ul ofsample was used for the initial dilution.

[0118] Assay Procedure

[0119] 250 ul of each standard working concentration, control and samplewas placed into two appropriately labeled microcentrifuge tubes. To eachtube 250 ul of 2% substrate solution was added with a repeater pipetteand a 12.5 ml Combitip set on 1. The samples were Vortexed for 3 secondsand incubated at 40° C. for 20 minutes. The samples were remove from thewater bath and immediately 1.0 ml of 95% EtOH was added to the samplesin the same order as above. A repeater pipette and a 12.5 or 50 mlCombitip set on 4 or 1, was used. The samples were vortexed for 3seconds. The samples were incubated at room temperature for 5-10minutes, then centrifuged for ten minutes in a benchtop centrifuge. Thesupernatant of the standards and samples were read in aspectrophotometer at 510 nm using 1.5 mL cuvettes. (Thespectrophotometer was zeroed with 95% EtOH)

[0120] Calculations

[0121] Using the standard concentrations and correlating absorbances(subtracting the blank absorbance), a calibration curve is developedwith a computer spreadsheet, programmable calculator, or graph paper.The curve should be linear over the range of the standard concentrationswith a correlation coefficient (r) of 0.998 or greater. The precision ofthe assay should fall between 5-10% CV. For liquids: u/ml=(u/ml fromstandard curve)*(sample dilution)

1 9 1 2794 DNA Bacillus deramificans misc_feature (1)...(2794) n = A, T,C, or G 1 gatgggaaca cgacaacgat cattgtccac tatttttgcc ctgctggtgattatcaacct 60 tggagtctat ggatgtggcc aaaagacgga ggtggggctg aatacgatttcaatcaaccg 120 gctgactctt ttggagctgt tgcaagtgct gatattccag gaaacccaagtcaggtagga 180 attatcgttc gcactcaaga ttggaccaaa gatgtgagcg ctgaccgctacatagattta 240 agcaaaggaa atgaggtgtg gcttgtagaa ggaaacagcc aaattttttataatgaaaaa 300 gatgctgagg atgcagctaa acccgctgta agcaacgctt atttagatgcttcaaaccag 360 gtgctggtta aacttagcca gccgttaact cttggggaag gnnnaagcggctttacggtt 420 catgacgaca cagcaaataa ggatattcca gtgacatctg tgaaggatgcaagtcttggt 480 caagatgtaa ccgctgtttt ggcaggtacc ttccaacata tttttggaggttccgattgg 540 gcacctgata atcacagtac tttattaaaa aaggtgacta acaatctctatcaattctca 600 ggagatcttc ctgaaggaaa ctaccaatat aaagtggctt taaatgatagctggaataat 660 ccgagttacc catctgacaa cattaattta acagtccctg ccggcggtgcacacgtcact 720 ttttcgtata ttccgtccac tcatgcagtc tatgacacaa ttaataatcctaatgcggat 780 ttacaagtag aaagcggggt taaaacggat ctcgtgacgg ttactctaggggaagatcca 840 gatgtgagcc atactctgtc cattcaaaca gatggctatc aggcaaagcaggtgatacct 900 cgtaatgtgc ttaattcatc acagtactac tattcaggag atgatcttgggaatacctat 960 acacagaaag caacaacctt taaagtctgg gcaccaactt ctactcaagtaaatgttctt 1020 ctttatgaca gtgcaacggg ttctgtaaca aaaatcgtac ctatgacggcatcgggccat 1080 ggtgtgtggg aagcaacggt taatcaaaac cttgaaaatt ggtattacatgtatgaggta 1140 acaggccaag gctctacccg aacggctgtt gatccttatg caactgcgattgcaccaaat 1200 ggaacgagag gcatgattgt ggacctggct aaaacagatc ctgctggctggaacagtgat 1260 aaacatatta cgccaaagaa tatagaagat gaggtcatct atgaaatggatgtccgtgac 1320 ttttccattg accctaattc gggtatgaaa aataaaggga agtatttggctcttacagaa 1380 aaaggaacaa agggccctga caacgtaaag acggggatag attccttaaaacaacttggg 1440 attactcatg ttcagcttat gcctgttttc gcatctaaca gtgtcgatgaaactgatcca 1500 acccaagata attggggtta tgaccctcgc aactatgatg ttcctgaagggcagtatgct 1560 acaaatgcga atggtaatgc tcgtataaaa gagtttaagg aaatggttctttcactccat 1620 cgtgaacaca ttggggttaa catggatgtt gtctataatc atacctttgccacgcaaatc 1680 tctgacttcg ataaaattgt accagaatat tattaccgta cgatgatccaggtaattata 1740 ccaacggatc aggtactgga aatgaaattg cangcngaaa ggccaatggttcaaaaattt 1800 attattgatt cccttaagta ttgggtcaat gagtatcata ttgacggcttccgttttgac 1860 ttaatggcgc tgcttggaaa agacacgatg tccaaagctg cctcggagcttcatgctatt 1920 aatccaggaa ttgcacttta cggtgagcca tggacgggtg gaacctctgcactgccagat 1980 gatcagcttc tgacaaaagg agctcaaaaa ggcatgggag tagcggtgtttaatgacaat 2040 ttacgaaacg cgttggacgg caatgtcttt gattcttccg ctcaaggttttgcgacaggt 2100 gcaacaggct taactgatgc aattaagaat ggcgttgagg ggagtattaatgactttacc 2160 tcttcaccag gtgagacaat taactatgtc acaagtcatg ataactacaccctttgggac 2220 aaaatagccc taagcaatcc taatgattcc gaagcggatc ggattaaaatggatgaactc 2280 gcacaagcag ttgttatgac ctcacaaggc gttccattca tgcaaggcggggaagaaatg 2340 cttcgtanaa aaggcggcaa cgacaatagt tataatgcag gcgatgcggtcaatgagttt 2400 gattggagca ggaaagctca atatccagat gttttcaact attatagcgggctaatccac 2460 cttcgtcttg atcacccagc cttccgcatg acgacagcta atgaaatcaatagccacctc 2520 caattcctaa atagtccaga gaacacagtg gcctatgaat taactgatcatgttaataaa 2580 gacaaatggg gaaatatcat tgttgtttat aacccaaata aaactgtagcaaccatcaat 2640 ttgccgagcg ggaaatgggc aatcaatgct acgagcggta aggtaggagaatccaccctt 2700 ggtcaagcag agggaagtgt ccaagtacca ggtatatcta tgatgatccttcatcaagag 2760 gtaagcccag accacggtaa aaagtaatag aaaa 2794 2 956 PRTBacillus deramificans VARIANT (1)...(956) Xaa = Any Amino Acid 2 Met AlaLys Lys Leu Ile Tyr Val Cys Leu Ser Val Cys Leu Val Leu 1 5 10 15 ThrTrp Ala Phe Asn Val Lys Gly Gln Ser Ala His Ala Asp Gly Asn 20 25 30 ThrThr Thr Ile Ile Val His Tyr Phe Cys Pro Ala Gly Asp Tyr Gln 35 40 45 ProTrp Ser Leu Trp Met Trp Pro Lys Asp Gly Gly Gly Ala Glu Tyr 50 55 60 AspPhe Asn Gln Pro Ala Asp Ser Phe Gly Ala Val Ala Ser Ala Asp 65 70 75 80Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg Thr Gln Asp 85 90 95Trp Thr Lys Asp Val Ser Ala Asp Arg Tyr Ile Asp Leu Ser Lys Gly 100 105110 Asn Glu Val Trp Leu Val Glu Gly Asn Ser Gln Ile Phe Tyr Asn Glu 115120 125 Lys Asp Ala Glu Asp Ala Ala Lys Pro Ala Val Ser Asn Ala Tyr Leu130 135 140 Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln Pro Leu ThrLeu 145 150 155 160 Gly Glu Gly Xaa Ser Gly Phe Thr Val His Asp Asp ThrAla Asn Lys 165 170 175 Asp Ile Pro Val Thr Ser Val Lys Asp Ala Ser LeuGly Gln Asp Val 180 185 190 Thr Ala Val Leu Ala Gly Thr Phe Gln His IlePhe Gly Gly Ser Asp 195 200 205 Trp Ala Pro Asp Asn His Ser Thr Leu LeuLys Lys Val Thr Asn Asn 210 215 220 Leu Tyr Gln Phe Ser Gly Asp Leu ProGlu Gly Asn Tyr Gln Tyr Lys 225 230 235 240 Val Ala Leu Asn Asp Ser TrpAsn Asn Ser Tyr Pro Ser Asp Asn Ile 245 250 255 Asn Leu Thr Val Pro AlaGly Gly Ala His Val Thr Phe Ser Tyr Ile 260 265 270 Pro Ser Thr His AlaVal Tyr Asp Thr Ile Asn Asn Pro Asn Ala Asp 275 280 285 Leu Gln Val GluSer Gly Val Lys Thr Asp Leu Val Thr Val Thr Leu 290 295 300 Gly Glu AspPro Asp Val Ser His Thr Leu Ser Ile Gln Thr Asp Gly 305 310 315 320 TyrGln Ala Lys Gln Val Ile Pro Arg Asn Val Leu Asn Ser Ser Gln 325 330 335Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr Thr Gln Lys Ala 340 345350 Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln Val Asn Val Leu 355360 365 Leu Tyr Asp Ser Ala Thr Gly Ser Val Thr Lys Ile Val Pro Met Thr370 375 380 Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn Gln Asn LeuGlu 385 390 395 400 Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly SerThr Arg Thr 405 410 415 Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro AsnGly Thr Arg Gly 420 425 430 Met Ile Val Asp Leu Ala Lys Thr Asp Pro AlaGly Trp Asn Ser Asp 435 440 445 Lys His Ile Thr Pro Lys Asn Ile Glu AspGlu Val Ile Tyr Glu Met 450 455 460 Asp Val Arg Asp Phe Ser Ile Asp ProAsn Ser Gly Met Lys Asn Lys 465 470 475 480 Gly Lys Tyr Leu Ala Leu ThrGlu Lys Gly Thr Lys Gly Pro Asp Asn 485 490 495 Val Lys Thr Gly Ile AspSer Leu Lys Gln Leu Gly Ile Thr His Val 500 505 510 Gln Leu Met Pro ValPhe Ala Ser Asn Ser Val Asp Glu Thr Asp Pro 515 520 525 Thr Gln Asp AsnTrp Gly Tyr Asp Pro Arg Asn Tyr Asp Val Pro Glu 530 535 540 Gly Gln TyrAla Thr Asn Ala Asn Gly Asn Ala Arg Ile Lys Glu Phe 545 550 555 560 LysGlu Met Val Leu Ser Leu His Arg Glu His Ile Gly Val Asn Met 565 570 575Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile Ser Asp Phe Asp 580 585590 Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Met Ile Gln Val Ile Ile 595600 605 Pro Thr Asp Gln Val Leu Glu Met Lys Leu Xaa Ala Glu Arg Pro Met610 615 620 Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Tyr Trp Val Asn GluTyr 625 630 635 640 His Ile Asp Gly Phe Arg Phe Asp Leu Met Ala Leu LeuGly Lys Asp 645 650 655 Thr Met Ser Lys Ala Ala Ser Glu Leu His Ala IleAsn Pro Gly Ile 660 665 670 Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly ThrSer Ala Leu Pro Asp 675 680 685 Asp Gln Leu Leu Thr Lys Gly Ala Gln LysGly Met Gly Val Ala Val 690 695 700 Phe Asn Asp Asn Leu Arg Asn Ala LeuAsp Gly Asn Val Phe Asp Ser 705 710 715 720 Ser Ala Gln Gly Phe Ala ThrGly Ala Thr Gly Leu Thr Asp Ala Ile 725 730 735 Lys Asn Gly Val Glu GlySer Ile Asn Asp Phe Thr Ser Ser Pro Gly 740 745 750 Glu Thr Ile Asn TyrVal Thr Ser His Asp Asn Tyr Thr Leu Trp Asp 755 760 765 Lys Ile Ala LeuSer Asn Pro Asn Asp Ser Glu Ala Asp Arg Ile Lys 770 775 780 Met Asp GluLeu Ala Gln Ala Val Val Met Thr Ser Gln Gly Val Pro 785 790 795 800 PheMet Gln Gly Gly Glu Glu Met Leu Arg Xaa Lys Gly Gly Asn Asp 805 810 815Asn Ser Tyr Asn Ala Gly Asp Ala Val Asn Glu Phe Asp Trp Ser Arg 820 825830 Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser Gly Leu Ile His 835840 845 Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr Ala Asn Glu Ile850 855 860 Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn Thr Val AlaTyr 865 870 875 880 Glu Leu Thr Asp His Val Asn Lys Asp Lys Trp Gly AsnIle Ile Val 885 890 895 Val Tyr Asn Pro Asn Lys Thr Val Ala Thr Ile AsnLeu Pro Ser Gly 900 905 910 Lys Trp Ala Ile Asn Ala Thr Ser Gly Lys ValGly Glu Ser Thr Leu 915 920 925 Gly Gln Ala Glu Gly Ser Val Gln Val ProGly Ile Ser Met Met Ile 930 935 940 Leu His Gln Glu Val Ser Pro Asp HisGly Lys Lys 945 950 955 3 718 PRT Bacillus subtilis 3 Met Val Ser IleArg Arg Ser Phe Glu Ala Tyr Val Asp Asp Met Asn 1 5 10 15 Ile Ile ThrVal Leu Ile Pro Ala Glu Gln Lys Glu Ile Met Thr Pro 20 25 30 Pro Phe ArgLeu Glu Thr Glu Ile Thr Asp Phe Pro Leu Ala Val Arg 35 40 45 Glu Glu TyrSer Leu Glu Ala Lys Tyr Lys Tyr Val Cys Val Ser Asp 50 55 60 His Pro ValThr Phe Gly Lys Ile His Cys Val Arg Ala Ser Ser Gly 65 70 75 80 His LysThr Asp Leu Gln Ile Gly Ala Val Ile Arg Thr Ala Ala Phe 85 90 95 Asp AspGlu Phe Tyr Tyr Asp Gly Glu Leu Gly Ala Val Tyr Thr Ala 100 105 110 AspHis Thr Val Phe Lys Val Trp Ala Pro Ala Ala Thr Ser Ala Ala 115 120 125Val Lys Leu Ser His Pro Asn Lys Ser Gly Arg Thr Phe Gln Met Thr 130 135140 Arg Leu Glu Lys Gly Val Tyr Ala Val Thr Val Thr Gly Asp Leu His 145150 155 160 Gly Tyr Glu Tyr Leu Phe Cys Ile Cys Asn Asn Ser Glu Trp MetGlu 165 170 175 Thr Val Asp Gln Tyr Ala Lys Ala Val Thr Val Asn Gly GluLys Gly 180 185 190 Val Val Leu Arg Pro Asp Gln Met Lys Trp Thr Ala ProLeu Lys Pro 195 200 205 Phe Ser His Pro Val Asp Ala Val Ile Tyr Glu ThrHis Leu Arg Asp 210 215 220 Phe Ser Ile His Glu Asn Ser Gly Met Ile AsnLys Gly Lys Tyr Leu 225 230 235 240 Ala Leu Thr Glu Thr Asp Thr Gln ThrAla Asn Gly Ser Ser Ser Gly 245 250 255 Leu Ala Tyr Val Lys Glu Leu GlyVal Thr His Val Glu Leu Leu Pro 260 265 270 Val Asn Asp Phe Ala Gly ValAsp Glu Glu Lys Pro Leu Asp Ala Tyr 275 280 285 Asn Trp Gly Tyr Asn ProLeu His Phe Phe Ala Pro Glu Gly Ser Tyr 290 295 300 Ala Ser Asn Pro HisAsp Pro Gln Thr Arg Lys Thr Glu Leu Lys Gln 305 310 315 320 Met Ile AsnThr Leu His Gln His Gly Leu Arg Val Ile Leu Asp Val 325 330 335 Val PheAsn His Val Tyr Lys Arg Glu Asn Ser Pro Phe Glu Lys Thr 340 345 350 ValPro Gly Tyr Phe Phe Arg His Asp Glu Cys Gly Met Pro Ser Asn 355 360 365Gly Thr Gly Val Gly Asn Asp Ile Ala Ser Glu Arg Arg Met Ala Arg 370 375380 Lys Phe Ile Ala Asp Cys Val Val Tyr Trp Leu Glu Glu Tyr Asn Val 385390 395 400 Asp Gly Phe Arg Phe Asp Leu Leu Gly Ile Leu Asp Ile Asp ThrVal 405 410 415 Leu Tyr Met Lys Glu Lys Ala Thr Lys Ala Lys Pro Gly IleLeu Leu 420 425 430 Phe Gly Glu Gly Trp Asp Leu Ala Thr Pro Leu Pro HisGlu Gln Lys 435 440 445 Ala Ala Leu Ala Asn Ala Pro Arg Met Pro Gly IleGly Phe Phe Asn 450 455 460 Asp Met Phe Arg Asp Ala Val Lys Gly Asn ThrPhe His Leu Lys Ala 465 470 475 480 Thr Gly Phe Ala Leu Gly Asn Gly GluSer Ala Gln Ala Val Met His 485 490 495 Gly Ile Ala Gly Ser Ser Gly TrpLys Ala Leu Ala Pro Ile Val Pro 500 505 510 Glu Pro Ser Gln Ser Ile AsnTyr Val Glu Ser His Asp Asn His Thr 515 520 525 Phe Trp Asp Lys Met SerPhe Ala Leu Pro Gln Glu Asn Asp Ser Arg 530 535 540 Lys Arg Ser Arg GlnArg Leu Ala Val Ala Ile Ile Leu Leu Ala Gln 545 550 555 560 Gly Val ProPhe Ile His Ser Gly Gln Glu Phe Phe Arg Thr Lys Gln 565 570 575 Gly ValGlu Asn Ser Tyr Gln Ser Ser Asp Ser Ile Asn Gln Leu Asp 580 585 590 TrpAsp Arg Arg Glu Thr Phe Lys Glu Asp Val His Tyr Ile Arg Arg 595 600 605Leu Ile Ser Leu Arg Lys Ala His Pro Ala Phe Arg Leu Arg Ser Ala 610 615620 Ala Asp Ile Gln Arg His Leu Glu Cys Leu Thr Leu Lys Glu His Leu 625630 635 640 Ile Ala Tyr Arg Leu Tyr Asp Leu Asp Glu Val Asp Glu Trp LysAsp 645 650 655 Ile Ile Val Ile His His Ala Ser Pro Asp Ser Val Glu TrpArg Leu 660 665 670 Pro Asn Asp Ile Pro Tyr Arg Leu Leu Cys Asp Pro SerGly Phe Gln 675 680 685 Glu Asp Pro Thr Glu Ile Lys Lys Thr Val Ala ValAsn Gly Ile Gly 690 695 700 Thr Val Ile Leu Tyr Leu Ala Ser Asp Leu LysSer Phe Ala 705 710 715 4 1091 PRT Klebsiella pneumonia 4 Met Leu ArgTyr Thr Arg Asn Ala Leu Val Leu Gly Ser Leu Val Leu 1 5 10 15 Leu SerGly Cys Asp Asn Gly Ser Ser Ser Ser Ser Ser Ser Gly Asn 20 25 30 Pro AspThr Pro Asp Asn Gln Asp Val Val Val Arg Leu Pro Asp Val 35 40 45 Ala ValPro Gly Glu Ala Val Thr Ala Val Glu Asn Gln Ala Val Ile 50 55 60 His LeuVal Asp Ile Ala Gly Ile Thr Ser Ser Ser Ala Ala Asp Tyr 65 70 75 80 SerSer Lys Asn Leu Tyr Leu Trp Asn Asn Glu Thr Cys Asp Ala Leu 85 90 95 SerAla Pro Val Ala Asp Trp Asn Asp Val Ser Thr Thr Pro Ser Gly 100 105 110Ser Asp Lys Tyr Gly Pro Tyr Trp Val Ile Pro Leu Asn Lys Glu Ser 115 120125 Gly Cys Ile Asn Val Ile Val Arg Asp Gly Thr Asp Lys Leu Ile Asp 130135 140 Ser Asp Leu Arg Val Ala Phe Gly Asp Phe Thr Asp Arg Thr Val Ser145 150 155 160 Val Ile Ala Gly Asn Ser Ala Val Tyr Asp Ser Arg Ala AspAla Phe 165 170 175 Arg Ala Ala Phe Gly Val Ala Leu Ala Glu Ala His TrpVal Asp Lys 180 185 190 Asn Thr Leu Leu Trp Pro Gly Gly Gln Asp Lys ProIle Val Arg Leu 195 200 205 Tyr Tyr Ser His Ser Ser Lys Val Ala Ala AspGly Glu Gly Lys Phe 210 215 220 Thr Asp Arg Tyr Leu Lys Leu Thr Pro ThrThr Val Ser Gln Gln Val 225 230 235 240 Ser Met Arg Phe Pro His Leu SerSer Tyr Ala Ala Phe Lys Leu Pro 245 250 255 Asp Asn Ala Asn Val Asp GluLeu Leu Gln Gly Glu Thr Val Ala Ile 260 265 270 Ala Ala Ala Glu Asp GlyIle Leu Ile Ser Ala Thr Gln Val Gln Thr 275 280 285 Ala Gly Val Leu AspAsp Ala Tyr Ala Glu Ala Ala Glu Ala Leu Ser 290 295 300 Tyr Gly Ala GlnLeu Ala Asp Gly Gly Val Thr Phe Arg Val Trp Ala 305 310 315 320 Pro ThrAla Gln Gln Val Asp Val Val Val Tyr Ser Ala Asp Lys Lys 325 330 335 ValIle Gly Ser His Pro Met Thr Arg Asp Ser Ala Ser Gly Ala Trp 340 345 350Ser Trp Gln Gly Gly Ser Asp Leu Lys Gly Ala Phe Tyr Arg Tyr Ala 355 360365 Met Thr Val Tyr His Pro Gln Ser Arg Lys Val Glu Gln Tyr Glu Val 370375 380 Thr Asp Pro Tyr Ala His Ser Leu Ser Thr Asn Ser Glu Tyr Ser Gln385 390 395 400 Val Val Asp Leu Asn Asp Ser Ala Leu Lys Pro Asp Gly TrpAsp Asn 405 410 415 Leu Thr Met Pro His Ala Gln Lys Thr Lys Ala Asp LeuAla Lys Met 420 425 430 Thr Ile His Glu Ser His Ile Arg Asp Leu Ser AlaTrp Asp Gln Thr 435 440 445 Val Pro Ala Glu Leu Arg Gly Lys Tyr Leu AlaLeu Thr Ala Gly Asp 450 455 460 Ser Asn Met Val Gln His Leu Lys Thr LeuSer Ala Ser Gly Val Thr 465 470 475 480 His Val Glu Leu Leu Pro Val PheAsp Leu Ala Thr Val Asn Glu Phe 485 490 495 Ser Asp Lys Val Ala Asp IleGln Gln Pro Phe Ser Arg Leu Cys Glu 500 505 510 Val Asn Ser Ala Val LysSer Ser Glu Phe Ala Gly Tyr Cys Asp Ser 515 520 525 Gly Ser Thr Val GluGlu Val Leu Asn Gln Leu Lys Gln Ser Asp Ser 530 535 540 Gln Asp Asn ProGln Val Gln Ala Leu Asn Thr Leu Val Ala Gln Thr 545 550 555 560 Asp SerTyr Asn Trp Gly Tyr Asp Pro Phe His Tyr Thr Val Pro Glu 565 570 575 GlySer Tyr Ala Thr Asp Pro Glu Gly Thr Thr Arg Ile Lys Glu Phe 580 585 590Arg Thr Met Ile Gln Ala Ile Lys Gln Asp Leu Gly Met Asn Val Ile 595 600605 Met Asp Val Val Tyr Asn His Thr Asn Ala Ala Gly Pro Thr Asp Arg 610615 620 Thr Ser Val Leu Asp Lys Ile Val Pro Trp Tyr Tyr Gln Arg Leu Asn625 630 635 640 Glu Thr Thr Gly Ser Val Glu Ser Ala Thr Cys Cys Ser AspSer Ala 645 650 655 Pro Glu His Arg Met Phe Ala Lys Leu Ile Ala Asp SerLeu Ala Val 660 665 670 Trp Thr Thr Asp Tyr Lys Ile Asp Gly Phe Arg PheAsp Leu Met Gly 675 680 685 Tyr His Pro Lys Ala Gln Ile Leu Ser Ala TrpGlu Arg Ile Lys Ala 690 695 700 Leu Asn Pro Asp Ile Tyr Phe Phe Gly GluGly Trp Asp Ser Asn Gln 705 710 715 720 Ser Asp Arg Phe Glu Ile Ala SerGln Ile Asn Leu Lys Gly Thr Gly 725 730 735 Ile Gly Thr Phe Ser Asp ArgLeu Arg Asp Ser Val Arg Gly Gly Gly 740 745 750 Pro Phe Asp Ser Gly AspAla Leu Arg Gln Asn Gln Gly Ile Gly Ser 755 760 765 Gly Ala Gly Val LeuPro Asn Glu Leu Ala Ser Leu Ser Asp Asp Gln 770 775 780 Val Arg His LeuAla Asp Leu Thr Arg Leu Gly Met Ala Gly Asn Leu 785 790 795 800 Ala AspPhe Val Met Ile Asp Lys Asp Gly Ala Ala Lys Lys Gly Ser 805 810 815 GluIle Asp Tyr Asn Gly Ala Pro Gly Gly Tyr Ala Ala Asp Pro Thr 820 825 830Glu Val Val Asn Tyr Val Ser Lys His Asp Asn Gln Thr Leu Trp Asp 835 840845 Met Ile Ser Tyr Lys Ala Ser Gln Glu Ala Asp Leu Ala Thr Arg Val 850855 860 Arg Met Gln Ala Val Ser Leu Ala Thr Val Met Leu Gly Gln Gly Ile865 870 875 880 Ala Phe Asp Gln Gln Gly Ser Glu Leu Leu Arg Ser Lys SerPhe Thr 885 890 895 Arg Asp Ser Tyr Asp Ser Gly Asp Trp Phe Asn Arg ValAsp Tyr Ser 900 905 910 Leu Gln Asp Asn Asn Tyr Asn Val Gly Met Pro ArgIle Ser Asp Asp 915 920 925 Gly Ser Asn Tyr Glu Val Ile Thr Arg Val LysGlu Met Val Ala Thr 930 935 940 Pro Gly Glu Ala Glu Leu Lys Gln Met ThrAla Phe Tyr Gln Glu Leu 945 950 955 960 Thr Glu Leu Arg Lys Ser Ser ProLeu Phe Thr Leu Gly Asp Gly Ser 965 970 975 Ala Val Met Lys Arg Val AspPhe Arg Asn Thr Gly Ser Asp Gln Gln 980 985 990 Ala Gly Leu Leu Val MetThr Val Asp Asp Gly Met Lys Ala Gly Ala 995 1000 1005 Ser Leu Asp SerArg Leu Asp Gly Leu Val Val Ala Ile Asn Ala Ala 1010 1015 1020 Pro GluSer Arg Thr Leu Asn Glu Phe Ala Gly Glu Thr Leu Gln Leu 1025 1030 10351040 Ser Ala Ile Gln Gln Thr Ala Gly Glu Asn Ser Leu Ala Asn Gly Val1045 1050 1055 Gln Ile Ala Ala Asp Gly Thr Val Thr Leu Pro Ala Trp SerVal Ala 1060 1065 1070 Val Leu Glu Leu Pro Gln Gly Glu Ala Gln Gly AlaGly Leu Pro Val 1075 1080 1085 Ser Ser Lys 1090 5 6 PRT ArtificialSequence Unknown 5 Asp Val Val Ile Asn His 1 5 6 9 PRT ArtificialSequence Unknown 6 Gly Phe Arg Leu Asp Ala Ala Lys His 1 5 7 6 PRTArtificial Sequence Unknown 7 Phe Val Asp Val His Asp 1 5 8 5 PRTArtificial Sequence Unknown 8 Tyr Asn Trp Gly Tyr 1 5 9 4 PRT ArtificialSequence Unknown 9 Val Trp Ala Pro 1

1. A modified pullulanase which is capable of catalyzing the hydrolysisof an alpha-1,6-glucosidic bond.
 2. The modified pullulanase of claim 1wherein said pullulanase is a modification of a pullulanase obtainablefrom a gram positive or a gram negative microorganism.
 3. The modifiedpullulanase of claim 2 wherein the gram positive microorganism includesB. subtilis, B. deramificans, B. stearothermophilus, B. naganoensis, B.flavocaldarius, B. acidopullulyticus, Bacillus sp APC-9603, B.sectorramus, B. cereus, and B. fermus.
 4. The modified pullulanase ofclaim 2 wherein the gram negative microorganism includes Klebsiellapneumonia and Klebsiella aerogenes.
 5. The modified pullulanase of claim3 wherein the B. deramificans pullulanase has the designation T89.117Din the LMG culture collection.
 6. The modified pullulanase of claim 1wherein the modification is a deletion of amino acids from the aminoterminus of about 100 amino acids.
 7. The modified pullulanase of claim1 wherein the modification is a deletion of amino acids from the aminoterminus of about 200 amino acids.
 8. The modified pullulanase of claim1 wherein the modification is a deletion of amino acids from the aminoterminus of about 300 amino acids.
 9. The modified pullulanase of claim6 wherein the deletion is 98 amino acids from the amino terminus of B.deramificans pullulanase.
 10. The modified pullulanase of claim 6wherein the deletion is 102 amino acids from the amino terminus of B.deramificans pullulanase.
 11. The modified pullulanase of claim 1wherein the modification is an addition of at least one amino acid tothe amino terminus of the mature pullulanase amino acid sequence. 12.The modified pullulanase of claim 11 wherein the pullulanase isobtainable from Bacillus deramificans and the additional amino acid atthe amino terminus is an Alanine.
 13. Modified pullulanase produced bythe method comprising the steps of obtaining a recombinant host cellcomprising nucleic acid encoding mature pullulanase, culturing said hostcell under conditions suitable for the production of modifiedpullulanase and optionally recovering the modified pullulanase.
 14. Themodified pullulanase of claim 13 wherein the nucleic acid encodingmature pullulanase has at least 70% identity to the polynucleotidesequence as shown in SEQ ID NO:1.
 15. The modified pullulanase of claim13 wherein the host cell is B. licheniformis which comprises a firstgene encoding Carlsberg protease and a second gene encoding endo Glu Cprotease, the first and/or second gene which codes for the protease(s)having been altered such that the protease activity is essentiallyeliminated.
 16. A nucleic acid comprising a polynucleotide sequenceencoding a modified pullulanase of claim
 1. 17. The nucleic acid ofclaim 16 having at least 70% identity to the polynucleotide sequenceshown in SEQ ID NO:
 1. 18. The nucleic acid of claim 16 having thepolynucleotide sequence as shown in SEQ ID NO:1.
 19. An expressionvector comprising the nucleic acid of claim
 16. 20. A host microorganismcomprising the expression vector of claim
 19. 21. The host microorganismof claim 20 wherein said microorganism is a Bacillus.
 22. The hostmicroorganism of claim 21 wherein said Bacillus includes B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and Bacillus thuringiensis.
 23. A method for the production of amodified pullulanase in a host cell comprising the steps of: a)obtaining a recombinant host cell comprising nucleic acid encoding amodified pullulanase; and b) culturing the microorganism underconditions suitable for the production of the modified pullulanase. 24.The method of claim 23 further comprising the step of: c) recovering themodified pullulanase.
 25. The method of claim 23 wherein the host cellis a Bacillus including B. subtilis, B. licheniformis, B. lentus, B.brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
 26. Themethod of claim 25 wherein the Bacillus host cell is B. licheniformis.27. An enzymatic composition comprising a modified pullulanase.
 28. Theenzymatic composition of claim 27 wherein the modified pullulanase has adeletion of amino acids from the amino terminus of up to about 100 aminoacids.
 29. The enzymatic composition of claim 27 wherein the modifiedpullulanase has a deletion of amino acids from the amino terminus of upto about 200 amino acids.
 30. The enzymatic composition of claim 27wherein the modified pullulanase has a deletion of amino acids from theamino terminus of up to about 300 amino acids.
 31. The composition ofclaim 27 wherein the modified pullulanase has the amino acid sequence asshown in SEQ ID NO:2 beginning at amino acid residue 99, a glutamicacid.
 32. The composition of claim 27 wherein the modified pullulanasehas the amino acid sequence as shown in SEQ ID NO:2 beginning at aminoacid residue 103, a glutamic acid.
 33. The composition of claim 27further comprising an enzyme selected from the group consisting ofglucoamylase, alpha-amylase, beta-amylase, alpha-glucosidase,isoamylase, cyclomaltodextrin, glucotransferase, beta-glucanase, glucoseisomerase, saccharifying enzymes, and/or enzymes which cleave glucosidicbonds.
 34. The composition of claim 27 further comprising aglucoamylase.
 35. The composition of claim 34 wherein the glucoamylaseis obtainable from an Aspergillus strain.
 36. The composition of claim35 wherein the Aspergillus strain includes Aspergillus niger,Aspergillus awamori and Aspergillus foetidus.
 37. The composition ofclaim 27 wherein said composition is in a solid form.
 38. Thecomposition of claim 27 wherein said composition is in a liquid form.39. The composition of claim 27 comprising at least 60% modifiedpullulanase.
 40. The composition of claim 27 comprising at least 80%modified pullulanase.
 41. A process for the saccharification of starch,wherein said process allows for reduced concentrations ofsaccharification by-products, comprising the step of contacting aqueousliquefied starch with an enzyme composition comprising modifiedpullulanase.
 42. The process of claim 41 wherein said modifiedpullulanase has a deletion of up to about 100 amino acids, up to about200 amino acids or up to about 300 amino acids from the amino terminusof pullulanase obtainable from a gram-negative or gram-positivemicroorganism.
 43. The process of claim 41 further comprising the stepsof heating said liquefied starch, and optionally recovering product. 44.The process of claim 41 wherein said enzyme composition furthercomprises glucoamylase.
 45. The process of claim 41 wherein said enzymecomposition comprises at least 80% modified pullulanase.
 46. The processof claim 41 wherein said contacting is at a pH of about less than orequal to 7.0 and greater than or equal to
 3. 47. The process of claim 41wherein the pH is about 4.2.
 48. The process of claim 41 wherein saidheating is at a temperature range of between 55 and 65 degrees C. 49.The process of claim 41 wherein the temperature is about 60 degrees C.50. B. licheniformis comprising nucleic acid encoding a modifiedpullulanase wherein said B. licheniformis comprises a first geneencoding Carlsberg protease and a second gene encoding endo Glu Cprotease, the first and/or second gene which codes for the protease(s)having been altered such that the protease activity is essentiallyeliminated.
 51. B. licheniformis comprising nucleic acid encoding amature pullulanase wherein said B. licheniformis comprises a first geneencoding Carlsberg protease and a second gene encoding endo Glu Cprotease, the first and/or second gene which codes for the protease(s)having been altered such that the protease activity is essentiallyeliminated.